Isolation, Characterisation of Polyhydroxyalkanoate Producing Bacteria from Oil Contaminated Site by SSR-IIJLS JOURNAL

Int. J. Life. Sci. Scienti. Res., VOL 2, ISSUE 2, pp: 102-109

(ISSN: 2455-1716)

Impact Factor 2.4

MARCH-2016

Research Article (Open access)

Isolation, Screening and Characterisation of Polyhydroxyalkanoate Producing Bacteria from Oil Contaminated Site Rameshwari, R1*, Meenakshisundaram, M2 1 2

Cauvery College For Women, Tiruchirapalli, T.N, India

Nehru Memorial College (Autonomous), Puthanampatti, India

ABSTRACT- The development of human civilization throughout history has led to growing disruption of the natural balance and the occurrence of different types of pollution. Environmental pollution with petroleum and petrochemical products has been recognized as significant and serious problem. Diesel engine oil, which is one of the major products of crude oil, constitutes a major source of pollution in our environment. Therefore diesel engine oil can enter into the environment through wrecks of oil tankers carrying diesel oil, cleaning of diesel tanks by merchants, war ships carrying diesel oil and motor mechanics. In present study the microorganisms utilising petrol and diesel oil as carbon source were isolated and investigation of their characteristics towards the production of polyhydroxyalkanoates (PHA), which is now a days well known as biodegradable polymer.

Key Words- Petrol and Diesel oil contamination, Bioremediation, Biodegradable bacterial polymer, Sudan Black B staining, 16sr RNA sequencing -------------------------------------------------IJLSSR-----------------------------------------------

INTRODUCTION Automobiles used (waste) oil contains oxidation products,

high volatile fractions, dissolution of water-soluble

sediments, water and metallic particles resulting from

fractions, photochemical oxidation, drill, emulsification,

machinery wears, used batteries, organic and inorganic

microbial degradation and sedimentation. The concentra-

chemicals used in oil additives and metals . Oil pollution

tion of hydrocarbon and non-hydrocarbon components in

occurs when oil is introduced into the environment directly

crude oil from different sources differ greatly2. The

or indirectly by men’s impacts resulting in unfavorable

contamination of the aquatic system with heavy metals has

change in such a way that safety and welfare of any living

been on the increase since the last century due to industrial

organisms is endangered. Crude oil if spilled into the water

activities. Heavy metals are taken up as cations. Among the

spreads over a wide area forming a slick and oil in water

heavy metals detected in WSF are Pb, Cu, Zn, Cd, Ni, Cr,

immediately begins to undergo a variety of physical,

and V 3. The extensive usage of petrochemical plastics due

chemical and biological changes including evaporation of

to their versatile properties especially durability is causing

severe problem in waste management affecting the

Address for Correspondence:

R. Rameshwari

aesthetic quality of cities, water bodies and natural areas.

Assistant Professor

The

Department of Biotechnology

concern in terms of the environment4. Biopolymers are one

Cauvery College For Women, Trichy–18, India Deepak

product that can help to overcome problems caused by

Prashar Received: 21 Jan 2016/Revised: 13 Feb 2016/Accepted: 26 Feb 2016

petrochemical polymers are generated from renewable

accumulation of plastic wastes has become a major

Page 102

Int. J. Life. Sci. Scienti. Res., VOL 2, ISSUE 2

natural sources and are often biodegradable and nontoxic5. The most extensively studied thermoplastic biopolymers are the polyhydroxyalkanoates (PHA) and polylactic acid LA)6. In the present study, was carried out physicochemical characterization of soils from auto-mechanic workshops at

from mechanic workshop Oil contaminated sediment sample from mechanic workshop

SS3

K.K.Nagar, Trichy (site 2)

Summer

different depths (0-15 cm; 15-30 cm, 30-45 cm), namely

Physiochemical analysis

pH, clay, silt, sand, total organic carbon (TOC), exchangea-

The physiochemical parameters, i.e., pH, electrical

ble cations, and equally examined the distribution of heavy

conductivity (EC) and total dissolved solids (TDS) were

metals such as Pb, Cr, Cu, Zn, Fe, Ni, and Cd.

measured using field kit (Thermo Orion 5-Star Ph

MATERIALS AND METHODS

Multi-Meter) on the site and the concentrations of soluble

Petroleum and Diesel oil contaminated water and sediment

cations and anions (Ca2+, Mg2+, Na+, K+, CO3−, HCO3−, Cl−

samples from various sources during post monsoon and

and SO42−) were determined

according to the standard

summer season as outlined in Table 1 and used for the

methods

physicochemical, Trace metal and bacteriological analysis

required for microbiological analysis, held on iceboxes and

and also used for the isolation of bacteria. The 2000 mL of

processed within 12 h of collection.

water samples were collected with a 2500 mL sterile

Trace Metal analysis

container in each location. Thesediment samples were

For heavy metal analysis, the one liter of oil polluted water

collected by sterile spatula and stored insterile plastic

was acidified immediately with concentrated nitric acid

8-11

. All samples were collected with precautions

bags .

(HNO3). For trace metal study, acidified test water samples

TABLE 1. Collection of samples from different sources

were filtered by Whatman No.1 filter paper and processed

for isolation of PHA Synthesizing bacteria

(APDC + MIBK) for metal analysis. The sediment samples

S. NO

SAMPLE TYPE

Oil contaminated water sample from mechanic workshop Oil contaminated water sample from mechanic workshop Oil contaminated water sample from mechanic workshop Oil contaminated sediment sample from mechanic workshop Oil contaminated sediment sample

SAMPLE CODE SW1

PLACE OF SAMPLING Woraiyur, Trichy (site 1)

COLLECTION TIME

Tennur, Trichy (site 3)

Summer

K.K.Nagar, Trichy (site 2)

Summer

were air-dried and smaller than (>) 63 μm in size were kept back in pre-cleaned properly. Thenceforth, the dried sediment samples were crushed by agate mortar and pestle. Both the samples were processed with an aqua-regia mixture (i.e. HCl: HNO3= 3:1) in Teflon bomb and were

SW2

incubated at 140 °C for 2-3 days after dried and sieved samples. After incubation, the reaction mixture was filtered with Whatman No.1 filter paper. The trace metals in the sea

SW3

water, sea sediment and crabsamples were determined by the atomic absorption spectrophotometry (GBC SensAA AAS, Australia) in flame mode12.

SS1

Woraiyur, Trichy (site 1)

Summer

Tennur, Trichy (site 3)

Summer

Isolation and Purification of Bacterial Strains 1 gm of oil contaminated soil sample was taken, serially diluted, plated on nutrient agar plates and incubated at

SS2

37°C for 24h to calculate the bacterial colonies13. By using the above method 1 ml of sago wastewater sample was

Page 103

Int. J. Life. Sci. Scienti. Res., VOL 2, ISSUE 2

taken for analysis and to calculate the bacterial colonies.

sequenced using the 518F/800R primers. Sequencing

After incubation all bacterial colonies were purified on

reactions were performed using a ABI PRISM® BigDyeTM

nutrient agar plates and subsequently analyzed for PHAs

Terminator Cycle Sequencing Kits with AmpliTaq® DNA

accumulation and confirmed by Nile blue A staining, gram

polymerase (FS enzyme) (Applied Biosystems). Amplified

reaction, motility, spores staining and biochemical tests.

DNA obtained from PCR was then sequenced by using

Nile Blue A Staining

aABI 3730xl sequencer (Applied Biosystems). The

Heat fixed bacterial smear was stained with 1% aqueous solution of Nile blue A at 55ºC for 10 min. The slide was washed with tap water to remove the excess stain and washed with 8% aqueous acetic acid for 1 min. The stained smear was again washed with tap water and blot dried. Prior to observation, the slide was remoistened with a drop of water and coverslip was placed on the smear14. The slides were viewed in fluorescence microscopy at a wavelength of 480 nm.

Morphological Characterisation The three potent PHA accumulating strains B5, B7 and B24 were examined for theircolony morphology, pigmentation fluorescence, cell shape and gram reaction as per thestandard procedures15.

Biochemical Characterisation Biochemical tests were carried out as per the method given by Cappuccino and Sherman (1992) with 24 hr old cultures.

sequence of 16s rRNA gene was compared with the 16s rRNA gene sequences available in the National Center for Biotechnolgy using

their

Information (NCBI) public databases by World

Wide

Web

and

the

BLAST

(Protein- Protein blast).

RESULTS AND DISCUSSION The physicochemical properties of petrol and diesel oil contaminated sediment and water samples in mechanic workshop for the season summer was given in Table 2. The development of human civilization throughout history has led to growing disruption of the natural balance and the occurrence of different types of pollution17. Changes in soil properties due to contamination with petroleum- derived substances can lead to water and oxygen deficits as well as shortage of available forms of nitrogen and phosphorus18-20. Studies have shown that PAHs can be carcinogenic and/or mutagenic in some circumstances and have been classified as priority pollutants21.

Molecular Characterisation The selected strain was identified by ABI 3730xl sequencer (Applied Biosystems). The 16S rRNA gene was selectively with the 16S rRNA gene universal primer.

Dna Extraction, Amplification And Sequencing Bacterial Genomic DNA was isolated using the Insta GeneTM Matrix Genomic DNA isolation kit. Extracted DNA was The 16s rRNA was amplified using bacterial universal primers for Bacillus sp. 27f forward primer and 1492R reverse primer. Amplified PCR product was

Page 104

Int. J. Life. Sci. Scienti. Res., VOL 2, ISSUE 2

Table 2. Physiochemical parameters in oil polluted area water and sediment samples in Tiruchirappalli, Tamil Nadu – Summer 2015 S. No.

Parameter

SW1

SW2

SW3

SS1

SS2

SS3

8.11

8.64

8.42

7.98

8.64

8.15

TDS (mg/L)

914.4

1070.6

800.8

1054.1

1255.8

1094

3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

EC (g/cm) Salinity (ppt) Do (mg/L) BOD (mg/L) TA (mg/L) TH (mg/L) Ca2+ (mg/L) Mg2+ (mg/L) Na+ (mg/L) K+ (mg/L) HCO3- (mg/L) CO3- (mg/L) Cl- (mg/L) SO42 (mg/L) N.NO2- (mg/L) OPO4- (mg/L)

1451.4 ~1 4.6 65.3 141 174.8 83.5 91.3 120.5 54.7 136.4 4.6 321.5 72.8 6.4 11.4

1699.3 ~1 3.5 70.1 202 156.9 72.4 84.5 104.2 71.6 185.6 16.4 400.8 81.3 14.5 25.9

1271.1 ~1 3.1 61.2 162.8 148 68.9 79.1 93.4 55.4 151.2 11.6 247.8 60.5 8.9 14.6

1673.1 ~1 2.8 89.4 171.5 222.7 101.4 121.3 108.9 64.8 160.1 11.4 365.2 80.5 10.5 12.4

1993.3 ~1 1.8 105.7 224.8 276.4 124.8 151.6 129.5 70.5 203.4 21.4 402.6 84.7 18.9 29.8

1736.5 ~1 2.2 91.5 203.1 239.1 108.4 130.7 115.8 76.8 186.7 16.4 334.5 76.5 12.4 21.3

Oil & Grease(mg/L)

11.3

13.4

9.4

17.6

18.6

14.5

According to Dorn et al. (1998), hydrocarbon contains substances that are toxic to the flora and fauna found in the ecosystem. Diesel pollution is on the increase in Nigeria, as well as other developing countries 23. The trace metal parameters of petrol and diesel oil contaminated soil and water samples in mechanic workshop for the season post-monsoon was given in Table 3. The presence of heavy metals in the environment and specifically in soils, industrial and domestic urban wastes endagers living organisms. Once it gets into food chain, through plants, animals and water sources leads to biomagnification and bioaccumulation in living cells and tissues24-25. Table 3. Trace metal parameters in oil polluted area water and sediment samples in Tiruchirappalli, Tamil Nadu – Summer 2015 S. No.

Parameter

SW1

SW2

SW3

SS1

SS2

SS3

2.12

4.1

2.41

3.31

4.69

2.96

0.68

1.12

0.57

0.95

1.21

1.03

2.78

4.23

3.18

4.87

5.58

4.38

8.92

10.56

7.45

15.65

18.94

16.54

0.31

0.52

0.35

0.72

0.42

2.14

2.64

1.87

3.12

4.12

3.64

4.56

5.98

4.12

7.45

9.26

7.95

Page 105

Int. J. Life. Sci. Scienti. Res., VOL 2, ISSUE 2

Totally 31 isolates were isolated from different samples from three different sites namely oil contaminated sites from mechanic workshop. All the strains were used for screening of PHA production.

SCREENING FOR PHA PRODUCING BACTERIAL STRAINS Totally five isolates from site 1 water sample (SW1) namely Acinetobacter sp, Aeromonas sp, Aeromonas sp, Alcaligenes sp and Bacillus sp among the isolates only one strain Bacillus sp showed PHA accumulation. In site 1 sediment sample (SS1) five bacterial strains were isolated such as Bacillus sp, Bacillus sp, Enterobacter sp, Lactobacillus sp, Listeria sp. From site 2 water sample (SW3) five bacterial strains were isolated namely Paenibacillus sp and from site 2 sediment sample (SS2) five isolates were identified. In site 3 water sample (SW3) five bacterial strains were isolated namely Pasteurella sp and different types of Pseudomonas sp were isolated and from sediment sample (SS3), six isolates were isolated such as Serratia sp, Citrobacter sp, staphylococcus sp and different types of Vibrio sp. These bacterial strains were identified and confirmed by morphological and biochemical test with the help of nineth editions of Bergeyâ&#x20AC;&#x2122;s manual of determinative bacteriology. The result of biochemical test was given on Table 4. Hence out of thiry one isolates one strain are screened for high PHA producers based on nile blue A staining (Fig. 1).

+ + +/-

+ + + + + + + + + + + + + + + + +/-

+ + + + + +/+ + + + + -

+/+/+/+/+ +/+/-

+/+ +/+ + + + + + + -

+ + + +/+/+ + +/+ + +

+ + + + + + + + + + -

+ + + + + + + + + + + + + + + + + + + + + + + + +

+ + + + + + + -

http://ijlssr.com IJLSSR ÂŠ 2016 All rights are reserved

+/+/+ +/+/+/+/+/+/+/+ + + +/+ + + +

+ + + + + + + + -

Cocci/ rods

+ + + + + + +/+ + + + + + + + + + + + + + +

L-Phenyl alanine Gram Staining

Sporulation

+ + + + + + + + + + +

Motility

+ + + + + + + + + + + + + + + +/-

Esculin

+ + + + + + + + + + + + + + +/+

ONPG

+ + + + + + + + + + + + + + + + + + + + + + + + + + + + +/+

Gelatin

+ + + +/+ + + + + -

Coagulase

+ + -

Casein Hydrolysis

Ak Ak Ac Ac Ak Ak Ac Ac Ac Ac Ac Ac Ac Ac Ac Ak Ak Ak Ak Ak Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac Ac

Nitrate

Urease

AK Ak Ac Ak Ak Ac Ac Ac Ac Ac Ak Ak Ak Ak Ak Ac Ak Ak Ak Ak Ak Ak Ak Ak Ak Ak Ac Ak Ak Ak Ak

Starch Hydrolysis Carbohydrate

Oxidase

+ + + + + + + + + + + + + + + + + + + + +

Catalase

Citrate

+ + + + + + + + +/+ + + + +

Gas

Voges Proskauer

+ + + + + + + +

H2S

Methyl Red

+ + + + + +/+

But

Indole

B1 B2 B3 B4 B5 B6 B7 B8 B9 B10 B11 B12 B13 B14 B15 B16 B17 B18 B19 B20 B21 B22 B23 B24 B25 B26 B27 B28 B29 B30 B31

TSI

Slant

Culture code

Table 4. Biochemical characterizations of isolated strains from oil polluted regions in Tiruchirappalli

Rods coccobacilli Rods Rods Rods Rods Rods Rods Rods coccobacilli Rods Rods Rods Rods Rods coccobacilli Rods Rods Rods Rods Rods Rods Rods Rods Rods Rods Rods Cocci coccobacilli Rods coccobacilli

Page 106

Int. J. Life. Sci. Scienti. Res., VOL 2, ISSUE 2

Fig 1. Nile blue A test for Bacillus spp.

Molecular Identification of Pure Isolate Using 16S rRNA The sequence of partial 16srRNA of this strain was compared against those available in the public database. The sequence is closely related to Bacillus sp (97% identity to 16srRNA sequence of similarity to strains). The nucleotide sequence and phylogeny based on these partial 16s rRNA sequences and related to bacterial strains are shown in Fig. 2 and Fig. 3. The

nucleotide sequence of

cgaaggcggc 721 tttttggtct gtaactgacg ctgaggcgcg aaagcgtggg gagcaaacag gattagatac 781 cctggtagtc cacgccgtaa acgatgagtg ctaagtgtta gagggtttcc gccctttagt 841 gctgcagcta acgcattaag cactccgcct ggggagtacg gtcgcaagac tgaaactcaa 901 aggaattgac gggggcccgc acaagcggtg gagcatgtgg tttaattcga agcaacgcga 961 agaaccttac caggtcttga catcctctga caactctaga gatagagcgt tccccttcgg 1021 gggacagagt gacaggtggt gcatggttgt cgtcagctcg tgtcgtgaga tgttgggtta 1081 agtcccgcaa cgagcgcaac ccttgatctt agttgccagc atttagttgg gcactctaag 1141 gtgactgccg gtgacaaacc ggaggaaggt ggggatgacg tcaaatcatc atgcccctta 1201 tgacctgggc tacacacgtg ctacaatgga tggtacaaag ggctgcaaga ccgcgaggtc 1261 aagccaatcc cataaaacca ttctcagttc ggattgtagg ctgcaactcg cctacatgaa 1321 gctggaatcg ctagtaatcg cggatcagca tgccgcggtg aatacgttcc cgggccttgt 1381 acacaccgcc cgtcacacca cgagagtttg taacacccga agtcggtgga gtaaccgtaa 1441 ggagctagcc gcctaaggtg ggacagatga ttggggtgaa gtcgtacggc taccccaaaa 1501 atgcccgcgg gagatgatcg tgatttcaag cggttctgga cactaaaacc ccctaccaga 1561 gaatgcttcg atacatcctc gccctcttcc gctcccagtc aagctccctt ctcctgttca 1621 gcctctcgct tgcatgtgtc gccgcacctc ctcgatgaaa cacgggcttta

16s rRNA of the strain Bacillus cereus determined in this study have been deposited the in Gen Bank database under accession number KU512626. ORIGIN 1 cgtaggatga cgctggcggc gtgcctaata catgcaagtc gagcgaactg attagaagct 61 tgcttctatg acgttagcgg cggacgggtg agtaacacgt gggcaacctg cctgtaagac 121 tgggataact tcgggaaacc gaagctaata ccggatagga tcttctcctt catgggagat 181 gattgaaaga tggtttcggc tatcacttac agatgggccc gcggtgcatt agctagttgg 241 tgaggtaacg gctcaccaag gcaacgatgc atagccgacc tgagagggtg atcggccaca 301 ctgggactga gacacggccc agactcctac gggaggcagc agtagggaat cttccgcaat 361 ggacgaaagt ctgacggagc aacgccgcgt gagtgatgaa ggctttcggg tcgtaaaact 421 ctgttgttag ggaagaacaa gtacgagagt aactgctcgt accttgacgg tacctaacca 481 gaaagccacg gctaactacg tgccagcagc cgcggtaata cgtaggtggc aagcgttatc 541 cggaattatt gggcgtaaag cgcgcgcagg cggtttctta agtctgatgt gaaagcccac 601 ggctcaaccg tggagggtca ttggaaactg gggaacttga gtgcagaaga gaaaagcgga 661 attccacgtg tagcggtgaa atgcgtagag atgtggagga acaccagtgg

CONCLUSION This study has led to the preliminary finding of bacterial spps. Bacillus cereusfrom oil contaminated mechanic workshop water sample capable of producing PHA. It was also observed that physicochemical and trace metal content of the site.

Page 107

Int. J. Life. Sci. Scienti. Res., VOL 2, ISSUE 2 [9] Vignesh S, Dahms HU, Emmanuel KV, Gokul MS,

ACKNOWLEDGEMENT One of authors (R. Rameshwari) is highly thankful to University Grants Commission (UGC), for financial support by receiving Minor Research Project (SERO/UGC, Hyderabad) No: MRP – 6112/15). Authors also thankful to Department of Civil Engineering, National Institute of Technolgy (NIT), Trichy, Tamilnadu, India for trace metal analysis.

Muthukumar K, Kim BR and James RA, Physicochemical parameters aid microbial community? A case study from marine recreational beaches, Southern India, Environmental Monitoring and Assessment, 186(3), 1875–1887 (2014). [10] Maiti S.K. 2003. Handbook of Methods in Environmentl studies. Vol. 2 Air, Noise, Soil and Overburden Analysis. Oxford Book Company, Jaipur, Rajasthan, India. [11] Osuji L.C., Nwoye I. 2007. An appraisal of the impact of

REFERENCES

petroleum

[1] European Environment Agency, EEA (2007) Progress in

hydrocarbonson

soil

fertility:

the

Owaza

experience. African J. Agric. Res. 2: 318–324.

Management of Contaminated Sites (CSI 015). EEA.

[12] Vignesh S, Muthukumar K and James RA, Antibiotic

Assessment Published July 2005; Kongen, ytorv, 6

resistant pathogens versus human impacts: A study from

DK-1050, Denmark.

three eco-regions of the Chennai coast, southern India,

[2] Brunnock, J.V., Duckworth, D.F. and Stephens, G.C. Analysis of Bench Pollutants. In: Hepple, P. (ed). Scientific Aspects of Pollution of the Sea by Oil. Institute of [3] Edema, N. E. Ionic and Physical Characteristics of the Water Soluble Fraction of Crude Oil and the Effects and of

Aquatic

Macrophytes.

Ph.D.

[13] Anonymous. Manual of microbiological methods Mc Graw Hill Book Company inc., New York, 1957; pp. 127. [14] Ostle, A.G Holt, J.G. Nile blue A a fluorescent stain for poly

Petroleum Printing Press. London, 1968; pp: 12-27.

Physiology

Marine Pollution Bulletin, 2012; 64, 790–800.

Thesis.

University of Benin, Benin City, 2006.

β hydroxybutyrate. Appl. Environ.Microbiol; 1982; 171: 73- 80. [15] Barthalomew, J. W. and Mittewer, T. A simplified bacterial strain. StainTechnology, 1950; 25: 153.

[4] Caesar, P and Archana, T. In Integration of natural and

[16] Cappuccino, T. G. and Sherman, N, Microbiology, a

Biological sources for the Production of Biopolymer: Actual

laboratory manual. The Benjamin/cummings publishing,

and Potential Utilization of various Wastes, Caesar Preeti et

Company Inc., California, 1992.

al. / J Phar. Res; 2010; 4(1): 53-55. [5] Sathesh Prabu, C and Murugesan, A.G. In “Effective

[17] Alexander,

estimation

Aging,

risk

bioavailability,

from

and

environmental

over

pollutants.

Utilization and Management of Coir Industrial waste for the

Environmental Science and Technology, 2000; 34(20):

Production of poly- β- hydroxybutyrate (PHB) using the

4259–4265.

Bacterium Azotobacter Beijerinickii, 2009. [6] Chen, G.Q. A microbial polyhydroxyalkanoates (PHA) based bio- and materials industry. Chem. Soc. Rev. 2009; 38:

[18] Wyszkowska,

and

Kucharski,

Biochemical

properties of soil contaminated by petrol. Polish Journal of Environmental Studies2000; 9(6): 476-485. [19] Ayotamuno, M. J., Kogbara, R. B. and Egwuenum, P. N.

2434–2446. [7] Vignesh S, Muthukumar K, Santhosh Gokul M and Arthur

Comparism

corn

and

elephant

grass

the

James R, Microbial pollution indicators in Cauvery river,

phytoremediation of a petroleum hydrocarbon contaminated

southern India. In Mu. Ramkumar (Ed.), On a Sustainable

agricultural soil in Port-Harcourt, Nigeria. Journal of

Future of the Earth’s Natural Resources, Springer earth

Agriculture and Environment, 2006; 4 (2&4): 218- 222.

system sciences, 2013; 363–376. [8] APHA (American Public Health Association), Standard

[20] Amellal N., Portal J.M., and Berthelin J., Effect of soil structure

bioavailability

polycyclic

aromatic

methods for the examination of water and wastewater. 19th

hydrocarbons within aggregates of a contaminated soil.

edn, Washington, DC, 1998.

Applied Geochemistry, 2001; 16:1611-19.

Page 108

Int. J. Life. Sci. Scienti. Res., VOL 2, ISSUE 2 [21] Dorn, P. B., Vipond, J. P., Salanitro, T. E. and Wisniewski,

[23] A.B. Rabah and M.L. Ibrahim, Physico-Chemical and

H. L. Assessment of the acute toxicity of crude oil in soils

Microbiological Characterization of Soils Lender with

using earthworms, microtoxic and plants.Chemosphere,

Tannery Effluents in Sokoto, Nigeria, Nig. J. Of Basic and

1998; 37: 845-860.

Applied Sc., 2010, 18 (1) 65-71, 30.

[22] Stephen, E., Okolo, M. O. and Akogu, E. A. Microbiological

[24] O.U. Ezeronye O.U. and A.O. Ubalua. Studies in the Effect

properties of oil impacted soil 36 months after diesel spill.

of Abattoir and Industrial Effluents on the Heavy Metals and

International Journal of Education, Science, Mathematics

Microbial Quality of Aba River Nigeria, Afr. J Biotechnol.,

and Environmental Studies, 2011; 3(1): 77-82.

2005, 4(3), 3266-272.

http://ijlssr.com IJLSSR ÂŠ 2016 All rights are reserved

Page 109